The goal of this proposal is to explore the structure of the RNA polymerase II transcription initiation complex using a combination of genetic and biochemical assays. The yeast Saccharomyces cerevisiae provides an ideal system for carrying out coordinated in vivo and in vitro studies that are not feasible in other model systems. The yeast transcription machinery is highly homologous to that in mammalian cells, so findings in one system can be extended to the other. The studies proposed here will contribute to our understanding of gene expression and of how defects in this process lead to cancer or developmental abnormalities. In addition, understanding the specific details of transcription in yeast may provide useful information for designing antifungal drugs. Genes for specific basal transcription will be mutagenized and introduced into yeast cells. Mutant genes that confer interesting phenotypes (e.g. high or low temperature sensitivity, inability to grow on certain media) will be further characterized. Currently, mutants have been isolated in the genes encoding the TATA Binding Protein (TBP), TFIIB, the Tfb1 subunit of TFIIH, the small subunit of TFIIF, and the large subunit of TFIIE. This analysis will be extended to the remaining subunits of TFIID (TAFs), TFIIE, TFIIF, and TFIIH. Many of the basal transcription factors interact with each other, so biochemical assays such as in vitro transcription, native gel electrophoresis, and co-immunoprecipitation will be used to assay protein-protein interactions with the mutant proteins. Defects in specific biochemical functions will be correlated with mutations in specific residues or regions of the transcription factors. It is expected that this approach will allow specific activities and protein- protein contacts to be studied. In cases where mutations in one transcription factor disrupt interactions with a second factor and thereby cause a conditional phenotype, suppressors in the second factor gene will be isolated that restore growth at the restrictive temperature. Again, the in vivo behavior of the mutant-suppressor pairs will be explored by testing protein-protein interactions in vitro. Also, random mutagenesis will be used to isolate second-site suppressors of mutant transcription factors. The proteins identified as suppressors (whether known factors or new proteins) will be characterized as to their interactions with the transcription machinery.